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  1. 1. + Isolation, Purification, and Assay of Wheat Germ Acid Phosphatase Natalia Manzano Wilmarie Morales RISE Program May 10th, 2012
  2. 2. + Introduction  Wheat germ is a very small part of a wheat kernel.  Wheat germ is very high in protein.  It contains around 28% protein and has more protein than can be found in most meat products.  The human body needs protein in order to repair tissue damage and to help minerals and nutrients reach our cells.  It contains more potassium and iron than any other food source.  It also has calcium, zinc, magnesium, and vitamins A, B1, B3 and E.  Vitamins B1 and 3 are very important to maintain energy levels and maintain healthy muscles and organs. Vitamin E is a very important antioxidant, prevents heart disease, and is needed to strengthen the body’s immune system.  
  3. 3. + Acid Phosphatase  Acid phosphatase is a type of enzyme used to free attached phosphate groups from other molecules during digestion.  It is basically a phosphomonoesterase, a phosphatase that acts on monoesters.  It is stored in lysosomes and functions when these fuse with endosomes.  Phosphatase enzymes are also used by soil microorganisms to access organically bound phosphate nutrients.  Some plant roots, exude carboxylates that perform acid phosphatase activity, helping to mobilise phosphorus in nutrient-deficient soils.
  4. 4. + Wheat Germ Acid Phosphatase• Acid phosphatase occurs in plants and animals, and has an optimal pH below 7.• Catalyzes the hydrolysis phosphate groups from macromolecules and smaller molecules that are stored in the wheat seed.
  5. 5. http://www.google.com.pr/imgres?imgurl=http://nutritioncheckup.com/blog/wp-content/uploads/2010/10/wheat-germ.jpg&imgrefurl=http://nutritioncheckup.com/blog/?p%3D123&h=511&w=535&sz=72&tbnid=3DIxRSBTgElMbM:&tbnh=94&tbnw=98&zoom=1&docid=q_1lvNRjniiFCM&sa=X&ei=xYusT9XhDpKy8AT5ip3TBA&ved=0CJUBEPUBMAQ&dur=112+ • The growing wheat embryo uses the freed phosphate in germination and growth.
  6. 6. + Objectives  To purify and isolate acid phosphatase from wheat germ.  Determine protein concentration using Bicinchoninic Acid protein assay.
  7. 7. + Materials and Methods Materials25 g Wheat germ MnCl2, 1.0 MH2O, prechilled to Sodium acetate4˚C buffer, 1.0M (pH 5.7)Cheesecloth (NH4)2SO4, saturated (pH 5.5) MethodsIce, crushed Pasteur PipetsBCA Kit Gloves, disposable Bicinchoninic Acid protein assayBSA standard, 1.0 Weighing trays (BCA)mg/ml SDS PAGECentrifuge, high BalancespeedIce bucket Spectrophotometer , visibleMagnetic stirrer MicroplateWaterbaths (30˚C  and 70˚C)
  8. 8. + Procedure I: Obtaining Fractions* All centrifugation was done Filter wheat germ at 2800Xg with cheese cloth at 4° for 20min Centrifuge filtrate (70ml)Discard Pellet Fraction I Pellet Add 1.26 ml of MnCl2 1.0M Discard Pellet Add Sodium Acetate Centrifuge Centrifuge Supernatant 62 ml Supernatant 25 ml Fraction II Fraction III
  9. 9. Pellet Add 33.48 ml of Ammonium Sulfate (buffer) (Saturated 35%) Centrifuge Supernatant 92 ml CentrifugeDiscard Pellet Add 46.92 ml of Supernatant Ammonium Sulfate Hot-cold water bath 21 ml (Saturated 57%) Centrifuge Supernatant 136 ml Fraction IV Pellet FractionVSuspend in dH2O Centrifuge FractionDiscard Pellet VI
  10. 10. + BCA Assay  BCA serves the purpose of Procedure reacting with complexes between copper ions and peptide bonds to produceProcedure II: a purple end product.BCA Assay  Estimate visually or measure with a standard spectrophotometer or plate reader (562nm).  (Stoscheck ,2000)
  11. 11. + Add Sample+ dH2O+BCA
  12. 12. +Conc.Of Abs Vol. ofworking Blan Abs- Conc. orba samplestd k Blank (mg/ml) nce added(mg/mL Stock) used 0.16 0.09 1X 0.068 0.200 2.3 1 2 4 0.42 0.09 1X 0.330 0.600 6.9 4 4 0.54 0.09 1X 0.446 1.000 11.5 0 4 1X/1 0.10 0.09 0.007 0.020 2.3 0.1 0 1 4 1X/1 0.12 0.09 0.032 0.060 6.9 0 6 4 1X/1 0.12 0.09 0.034 0.100 11.5 0 8 4 1X/1 0.07 0.09 0.000 0.002 2.3 0.01 00 8 4 1X/1 0.08 0.09 0.000 0.006 6.9 00 3 4 Standard Concentration Curve 1X/1 0.09 0.09 0.000 0.010 11.5 00 4 4
  13. 13. + Procedure III: SDS-PAGE  Running Gel:  Stacking Gel:  1.88 ml dH2O  2.975 ml dH2O  1.67 ml separating buffer  1.25 ml separating buffer  2.22 ml Acrylamide  0.662 ml Acrylamide  50.0 µl 20% SDS  225.0 µl 20% SDS  100 µl 10% Glycerol  25 µl 10% Glycerol  1.67 µl TEMED  6.25 µl TEMED  50 µl APS(100 mg/ml)  25 µl APS
  14. 14. + Procedure III: SDS Got sample obtained from previous purifying technique (Purification)Set up gel, remove Loaded BuffercombStain and look at with Load Sample (3:1 Ran Gel sample:buffer)UV light
  15. 15. + Results
  16. 16. Protein Concentration Table Sample Absorbance Blank Abs-Blank Conc. (mg/ml) Vol. of sample added Dilution factor Fraction I 1X-L 2.511 0.094 2.417 25.466 2.3 1 Fraction I 1X-H 3.029 0.094 2.935 6.185 11.5 1 Fraction I X/10-L 0.305 0.094 0.211 22.231 2.3 10Fraction I X/10-H 1.171 0.094 1.077 22.695 11.5 10Fraction I X/100-L 0.13 0.094 0.036 37.930 2.3 100Fraction I X/100-H 0.241 0.094 0.147 30.976 11.5 100 Fraction II 1X-L 3.029 0.094 2.935 30.923 2.3 1 Fraction II 1X-H 3.029 0.094 2.935 6.185 11.5 1 Fraction II X/10-L 0.385 0.094 0.291 30.660 2.3 10 Fraction II X/10-H 1.636 0.094 1.542 32.493 11.5 10Fraction II X/100-L 0.164 0.094 0.07 73.752 2.3 100Fraction II X/100-H 0.512 0.094 0.418 88.081 11.5 100 Fraction III 1X-L 0.524 0.094 0.43 4.530 2.3 1 Fraction III 1X-H 2.035 0.094 1.941 4.090 11.5 1 Fraction III X/10-L 0.143 0.094 0.049 5.163 2.3 10Fraction III X/10-H 0.317 0.094 0.223 4.699 11.5 10Fraction III X/100-L 0.094 0.094 0 0.000 2.3 100Fraction III X/100-H 0.138 0.094 0.044 9.272 11.5 100 Fraction IV 1X-L 0.536 0.094 0.442 4.657 2.3 1 Fraction IV 1X-H 1.541 0.094 1.447 3.049 11.5 1 Fraction IV X/10-L 0.149 0.094 0.055 5.795 2.3 10 Fraction IV X/10-H 0.303 0.094 0.209 4.404 11.5 10Fraction IV X/100-L 0.102 0.094 0.008 8.429 2.3 100Fraction IV X/100-H 0.163 0.094 0.069 14.540 11.5 100 Fraction V 1X-L 2.373 0.094 2.279 24.012 2.3 1 Fraction V 1X-H 3.029 0.094 2.935 6.185 11.5 1 Fraction V X/10-L 0.179 0.094 0.085 8.956 2.3 10Fraction V X/10-H 0.673 0.094 0.579 12.201 11.5 10Fraction V X/100-L 0.151 0.094 0.057 60.055 2.3 100Fraction V X/100-H 0.358 0.094 0.264 55.630 11.5 100 Fraction VI 1X-L 1.443 0.094 1.349 14.213 2.3 1 Fraction VI 1X-H 3.029 0.094 2.935 6.185 11.5 1 Fraction VI X/10-L 0.212 0.094 0.118 12.432 2.3 10 Fraction VI X/10-H 0.657 0.094 0.563 11.864 11.5 10Fraction VI X/100-L 0.152 0.094 0.058 61.109 2.3 100Fraction VI X/100-H 0.333 0.094 0.239 50.362 11.5 100
  17. 17. Average Concentration Table Total Average conc. Samples (mg/mL) Volumes (mL) protein (mg) Fraction I 30.39 65 1974.62 Fraction II 64.16 62 3978.18 Fraction III 5.92 25 147.90 Fraction IV 7.56 21 158.86 Fraction V 34.21 136 4652.61 Fraction VI 33.94 78 2647.45      
  18. 18. + Expected Concentration Curve Fraction Concentration Curve
  19. 19. + Expected Trend Experimental Trend
  20. 20. Fraction VI Fraction V Fraction V Fraction III Fraction II Fraction I 250- 150- 100- 75- 50- 37- 25- 15- PAGE SDS-+
  21. 21. + Gel Results Expected Obtained
  22. 22. + Conclusion  Our results were not what was expected.  We were not able to successfully isolate wheat germ acid phosphatase.  This could be accountable to human error.  Most likely our errors in this experiment were calculating an incorrect volumes for ammonium sulfate and MnCl2 solutions that were added.  Another error could have been incorrectly prepare dilutions of the enzyme for the assay.
  23. 23. + Acknowledgements RISE Program Vibha Bansal PhD Edmarie Martinez Vivian Rodriguez Cruz Alexandra Rosado Burgos
  24. 24. + References 1. Stoscheck ,2000. CM. Quantitation of Protein. Methods in Enzymology, 50-69. 2. Yasuaki Kawarasaki, Hideo Nakano, Tsuneo Yamane, 1999. Purification and some properties of wheat germ acid phosphatases. Elsvier [http://www.sciencedirect.com/science/article/pii/016894529604 4779] 3. Ke-Xue Zhu,Hui-Ming Zhou, and Hai-Feng Qian, 2008. Proteins Extracted from Defatted Wheat Germ: Nutritional and Structural Properties [http://cerealchemistry.aaccnet.org/doi/abs/10.1094/CC-83-0069 ] 4. VERJEE Z. H. M., 1969. Isolation of Three Acid Phosphatases from Wheat Germ. European J. Biochem., 439-44.
  25. 25. + Questions?

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